Use of pak inhibitor for the treatment of a joint disease

ABSTRACT

The invention refers to the use of a p21-activated kinase (PAK) inhibitor as a target protein for the discovery of a PAK inhibitor as a medicament for the treatment of a joint disease.

The present invention refers to the use of a p21-activated kinase (PAK)inhibitor for the treatment of a joint disease such as osteoarthritis orrheumatoid arthritis or for the treatment of a joint pain and the use ofPAK as a target protein for the discovery of a PAK inhibitor as amedicament for the treatment of a joint disease.

Osteoarthritis is the most common disabling condition of man in thewestern world. Due to the aging of the population we have to face anever-increasing population of patients, whose quality of life isseverely affected. In addition, the disease carries a tremendoussocioeconomic burden with high direct and indirect costs. The currenttreatment modalities concentrate on the management of the painassociated with osteoarthritis, but we are still completely lacking anypharmacological treatment modalities able to slow, stop or even reversethe course of the disease.

Osteoarthritis can be viewed as the clinical and pathological outcome ofa range of disorders that results in structural and functional failureof synovial joints. Osteoarthritis occurs when the dynamic equilibriumbetween the breakdown and repair of joint tissues is overwhelmed.Structural failure of articular cartilage can result from abnormalmechanical strains injuring healthy cartilage, as well as from failureof pathologically impaired cartilage degenerating under the influence ofphysiological mechanical strains. Morphological changes observed inosteoarthritis include cartilage erosion as well as a variable degree ofsynovial inflammation. These changes are attributed to a complex networkof biochemical factors, including proteolytic enzymes, that lead to abreakdown of the cartilage macromolecules. Cytokines such as IL-1 andTNFα which are produced by activated synoviocytes, mononuclear cells orby articular cartilage itself, significantly upregulatemetalloproteinases (MMP) and cytokine gene expression, and bluntcompensatory synthesis pathways. For example, activation of theAP1-transcription factor complex by IL-1 and/or TNFα through signaltransmission via MAPK (mitogene activated protein kinase)-pathways playsan important role for the regulation of expression of marker genes thatare relevant for osteoarthritis.

A further biochemical factor involved in cartilage catabolism andgenesis of inflammatory pain is PGE2 (prostaglandin E2). PGE2, aneicosanoid synthesized by cycloxygenase (COX)-1 and -2, is involved inIL-1-mediated proteoglycan degradation, and administration of PGE2 intoconscious rats or mice induces hyperalgesia. Endogenous expression ofIL-1 leads to induction of COX-2 in the human osteoarthritis joint.

Consequently, osteoarthritis is characterized by a slow progressivedegeneration of articular cartilage. The exact etiology ofosteoarthritis is not yet known, but the degradation of cartilage matrixcomponents is generally agreed to be due to an increased synthesis andactivation of extracellular proteinases, mainly matrixmetalloproteinases, and cytokines that amplify degenerative processes.Novel approaches to treat osteoarthritis are required, and progress inunderstanding the biology of cartilage disorders has led to the use ofgenes whose products stimulate cartilage repair or inhibit breakdown ofthe cartilaginous matrix. Several studies illustrate e.g. the potentialimportance of modulating IL-1 activity as a means to reduce theprogression of the structural changes in osteoarthritis.

Therefore, an object of the present invention is to find new therapeuticways for the avoidance or reduction of the effects of the cartilageharming factors.

Surprisingly, PAK, in particular PAK1 has been identified as animportant mediator of the IL-1 induced activation of signalling pathwaysleading to an upregulation of the expression of marker genes that arerelevant for osteoarthritis.

PAK1 belongs to a member of the evolutionarily conserved family ofserine/threonine kinases that are important for a variety of cellularfunctions including cell morphogenesis, motility, survival, mitosis, andangiogenesis. PAK's belong to the larger family of Step 20 proteinkinases. Step 20p is a putative yeast mitogen-activated protein kinasekinase kinase kinase (MAP4K) involved in the mating pathway in S.cerevisiae. Its homologs in mammals, Drosophila, Caenorhabditis elegansand other organisms make up a large emerging group of protein kinasesincluding members in human. The Step 20 group kinases are furtherdivided into the p21-activated kinase (PAK) and germinal center kinase(GCK) families. They are characterized by the presence of a conservedkinase domain and a noncatalytic region of great structural diversitythat enables the kinases to interact with various signalling moleculesand regulatory proteins of the cytoskeleton.

Several publications have described a role for PAK's in the regulationof MAPK activity in mammalian cells (see e.g. Dan, C. et al. (2002) Mol.Cell Biol., 22, 567-577). MAPK cascades are crucial in a wide range ofcellular events, transmitting signals from extracellular stimuli such asgrowth factors, cytokines and environmental stresses to activatetranscription factors, resulting in regulation of gene expression(Johnson, G. L. and Lapadat, R. (2002) Science, 298, 1911-1912).Signalling is mediated by linear sequential phosphorylation of atriple-kinase module consisting of MAP kinase kinase kinase (MAP3K), MAPkinase kinase (MAP2K) and MAPK. The triple-kinase module and itsactivation mechanism are highly conserved in the eukaryotic evolutionfrom yeast to mammals.

In mammalian cells PAKs are identified as downstream effector target ofCdc42 and Rac1, and binding of GTPases to Pak1 stimulates its kinaseactivity via autophosphorylation. PAKs form complexes specifically withactivated (GTP-bound) p21, inhibiting p21 GTPase activity and leading tokinase autophosphorylation and activation. PAK family kinases, conservedfrom yeasts to humans, are directly activated by Cdc42 or Rac1 throughinteraction with a conserved N-terminal motif (corresponding to residues71 to 137 in a PAK). Autophosphorylated kinase has a decreased affinityfor Cdc42/Rac1, freeing the p21 for further stimulatory activities ordownregulation by GTPase-activating proteins (Manser, E. et al. (1994)Nature, 367, 40-46). In addition to Rac1 and Cdc42, newly identifiedhomologs of the Rho family of GTPases such as Wrch-1 and Chp can alsoactivate PAKs and induce filopodium formation and stress fiberdissolution (Aronheim, A. et al. (1998) Curr. Biol. 8, 1125-1128).Guanine nucleotide exchange factors (GEFs) and GTPase-activatingproteins (GAPs), which regulate the GTP-GDP bound states of the Rhofamily of GTPases, are important determinants of downstream signallingactivated by PAK1 kinases (Zhou, K. et al. (1998) J. Biol. Chem., 273,16782-16786).

The nucleic acid sequence and the amino acid sequence of PAK1 are shownin SEQ ID NO: 1 and 2, respectively. The sequences of PAK1 required fortight binding to Cdc42 and Rac have been studied by analyzing propertiesof truncated fragments and site-directed mutants as well as bydetermining the solution structure of a complex of Cdc42 with thehomologous segment of WASP (Burbelo, P. D. et al. (1995) J. Biol. Chem.,270, 29071-29074; Rudolph, M. G. et al. (1998) J. Biol. Chem., 273,18067-18076; Abdul-Manan, N. et al. (1999) Nature, 399, 379-383).Overlapping but not coincident with the PBD (p21-binding-domain) of PAK1is a segment implicated in autoinhibition (Zhao, Z. S. et al. (1998)Mol. Cell Biol, 18, 2153-2163; Lei, M. et al. (2000) Cell, 102,387-397). This autoregulatory region includes the inhibitory switch andkinase inhibitory domains, that interfere with kinase autoactivation(Lei, M. et al. (2000) Cell 102, 387-397). Mutations within theautoregulatory region yield constitutively-active mutants (Zhao, Z. S.et al. (1998), supra; Lei, M. et al. (2000) supra). Expression of theautoregulatory domain including aminoacids 83-149 (PID for PAKinhibitory domain) of PAK1 in mammalian cells prevents the activation ofdownstream effectors through PAK1. Thus, coexpression of this PAKinhibitor with the constitutively-active GTPase Cdc42^(G12V), anactivator of PAK1, prevented e.g. the formation of peripheral actinmicrospikes and associated loss of stress fibers normally induced bythis p21-protein (Zhao, Z. S. et al. (1998), supra).

In a recent reports, inhibition of PAK1 activity in breast cancer cellswas associated with a reduction in c-Jun N-terminal kinase activity,inhibition of DNA binding activity of transcription factor AP-1 andsuppression of in vivo transcription driven by AP-1 promoter which isknown to be involved in breast cancer invasion (Adam, L. et al. (2000)J. Biol. Chem., 275, 12041-12050). Furthermore, Ngo, the etiologic agentof gonorrhea, induces the activation of proinflammatory cytokines via acascade of cellular stress response kinases involving PAK, which directsthe signal from the Rho family of small GTPases to JNK and AP-1activation (Naumann, M. et al. (1998) J. Exp. Med., 188, 1277-1286).However, there has been no report about a potential role for PAK familykinases in a joint disease such as osteoarthritis.

One subject matter of the present invention is, therefore, the use of aPAK inhibitor for the treatment of a joint disease, in particular adegenerative joint disease such as osteoarthritis and/or an inflammatoryjoint disease such as rheumatoid arthritis. The PAK inhibitor can alsobe used to treat the joint pain, in particular by reducing the jointpain in degenerative joint diseases. In addition, a PAK inhibitor can beused for the production of a medicament for the treatment of a jointdisease and/or for the treatment of the joint pain as specified above.

According to the present invention the term “inhibitor” refers to abiochemical or chemical compound which preferably inhibits or reducesthe serine/threonine kinase activity of PAK or the expression of the PAKgene or the localization of PAK in the cell, as e.g. described inKiosses, W. B. et al. (2002) Circ. Research, Apr. 5, 2002, 697-702. Theserine/threonine kinase activity can be measured according to standardprotocols, e.g. with the HitHunter™ Serine/Threonine Kinase Assay ofApplied Biosystems, Inc., Foster City, Calif., U.S.A. The expression ofthe PAK gene can be measured by RT-PCT or Western blot analysis asdescribed in the examples of the present invention.

The term “PAK” refers to a family of serine/threonine p21-activatingkinases including, without limitation, PAK1, PAK2, PAK3 and/or PAK-4.These proteins preferably serve as targets for the small GTP bindingproteins Cdc42 and Rac as described above. In particular, the term “PAK”refers to human PAK, especially human PAK1. The nucleic acid and aminoacid sequences of human PAK1 are shown in SEQ ID NO: 1 and 2,respectively. The amino acid sequences of human PAK 2, 3 and 4 are shownin SEQ ID NO: 3, 4 and 5, respectively. The gene sequences coding forthese human PAKs can easily be derived by using the genetic code. Thegene bank accession numbers for human PAK 1, 2, 3 and 4 areNP_(—)002567, Q13177, NP_(—)002569 and NP_(—)005875, respectively. Thenon-human homologs can be isolated by means of the human PAK 1, 2, 3 or4 gene sequences with methods known to a person skilled in the art, e.g.through PCR amplification or hybridization under stringent conditions(e.g. 60° C. in 2.5×SSC buffer followed by several washing steps at 37°C. in a lower buffer concentration) with suitable probes derived frome.g. the human PAK sequences according to standard laboratory methods.

Examples of such PAK inhibitors are the PAK1 inhibitor domain with theamino acid sequence HTIHVGFDAV TGEFTGMPEQ WARLLQTSNI TKSEQKKNPQAVLDVLEFYN SKKTSNSQKY MSFTDKS (SEQ ID NO: 6), the PAK1 peptide with theamino acid sequence KPPAPPMRNT STM (SEQ ID NO: 7), the Tat-PAK fusionpeptide with the amino acid sequence YGRKKRRQRR RGKPPAPPMR NTSTM (SEQ IDNO: 8), binding proteins or binding peptides directed against PAK, inparticular against the active site of PAK, nucleic acids directedagainst the PAK gene or PAK itself, a chemical molecule, preferably asmall molecule, and/or a natural product extract.

According to the present invention the term “chemical molecule”encompasses non-polymeric organic compounds, lipids, carbohydrates,peptides, preferably peptides with about 10 to about 80 amino acids, inparticular with 10 to 25 amino acids and oligonucleotides, preferablywith about 10 to about 90 nucleotides, in particular with 15 to 25nucleotides. Especially preferred are small chemical molecules, inparticular non-polymeric organic compounds, either synthesized in alaboratory or found in nature, with a preferred molecular weight ofabout 200 g/mole to about 1500 g/mole, in particular 400 g/mole to 1000g/mole.

Alternatively the inhibitor of the present invention can be in the formof a natural product extract, either in crude or in purified form. Theextract can be produced according to standard procedures, such as waterand/or alcohol and/or organic solvent extraction and/or columnchromatography and/or precipitation from an animal, plant or microbialsource, like snake poison, leaves or microbial fermentation broths.

The term “binding protein” or “binding peptide” refers to a class ofproteins or peptides which bind and inhibit RAK including, withoutlimitation, polyclonal or monoclonal antibodies, antibody fragments andprotein scaffolds directed against PAK, e.g. anticalins which aredirected against PAK.

The procedure for preparing an antibody or antibody fragment is effectedin accordance with methods which are well known to the skilled person,e.g. by immunizing a mammal, for example a rabbit, with PAK, whereappropriate in the presence of, for example, Freund's adjuvant and/oraluminium hydroxide gels (see, for example, Diamond, B. A. et al. (1981)The New England Journal of Medicine: 1344-1349). The polyclonalantibodies which are formed in the animal as a result of animmunological reaction can subsequently be isolated from the blood usingwell known methods and, for example, purified by means of columnchromatography. Monoclonal antibodies can, for example, be prepared inaccordance with the known method of Winter & Milstein (Winter, G. &Milstein, C. (1991) Nature, 349, 293-299).

According to the present invention the term antibody or antibodyfragment is also understood as meaning antibodies or antigen-bindingparts thereof, which have been prepared recombinantly and, whereappropriate, modified, such as chimaeric antibodies, humanizedantibodies, multifunctional antibodies, bispecific or oligospecificantibodies, single-stranded antibodies and F(ab) or F(ab)₂ fragments(see, for example, EP-B1-0 368 684, U.S. Pat. No. 4,816,567, U.S. Pat.No. 4,816,397, WO 88/01649, WO 93/06213 or WO 98/24884).

As an alternative to the classical antibodies it is also possible, forexample, to use protein scaffolds against PAK, e.g. anticalins which arebased on lipocalin (Beste et al. (1999) Proc. Natl. Acad. Sci. USA, 96,1898-1903). The natural ligand-binding sites of the lipocalins, forexample the retinol-binding protein or the bilin-binding protein, can bealtered, for example by means of a “combinatorial protein design”approach, in such a way that they bind to selected haptens, here to PAK(Skerra, 2000, Biochim. Biophys. Acta, 1482, 337-50). Other knownprotein scaffolds are known as being alternatives to antibodies formolecular recognition (Skerra (2000) J. Mol. Recognit., 13, 167-187).

The term “nucleic acids against the PAK gene or PAK itself” refers todouble-stranded or single stranded DNA or RNA which, for example,inhibit the expression of the PAK gene or the activity of PAK andincludes, without limitation, antisense nucleic acids, aptamers, siRNAs(small interfering RNAs) and ribozymes.

The nucleic acids, e.g. the antisense nucleic acids, can be synthesizedchemically, e.g. in accordance with the phosphotriester method (see, forexample, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).Aptamers are nucleic acids which bind with high affinity to apolypeptide, here PAK. Aptamers can be isolated by selection methodssuch as SELEX (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50; Klugand Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; U.S. Pat. No.5,582,981) from a large pool of different single-stranded RNA molecules.Aptamers can also be synthesized and selected in their mirror-imageform, for example as the L-ribonucleotide (Nolte et al. (1996) Nat.Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14,1112-5). Forms which have been isolated in this way enjoy the advantagethat they are not degraded by naturally occurring ribonucleases and,therefore, possess greater stability.

Nucleic acids may be degraded by endonucleases or exonucleases, inparticular by DNases and RNases which can be found in the cell. It is,therefore, advantageous to modify the nucleic acids in order tostabilize them against degradation, thereby ensuring that a highconcentration of the nucleic acid is maintained in the cell over a longperiod of time (Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94;WO 95/11910; WO 98/37240; WO 97/29116). Typically, such a stabilizationcan be obtained by introducing one or more internucleotide phosphorusgroups or by introducing one or more non-phosphorus internucleotides.

Suitable modified internucleotides are compiled in Uhlmann and Peyman(1990), supra (see also Beigelman et al. (1995) Nucleic Acids Res.23:3989-94; WO 95/11910; WO 98/37240; WO 97/29116). Modifiedinternucleotide phosphate radicals and/or non-phosphorus bridges in anucleic acid which can be employed in one of the uses according to theinvention contain, for example, methyl phosphonate, phosphorothioate,phosphoramidate, phosphorodithioate and/or phosphate esters, whereasnon-phosphorus internucleotide analogues contain, for example, siloxanebridges, carbonate bridges, carboxymethyl esters, acetamidate bridgesand/or thioether bridges. It is also the intention that thismodification should improve the durability of a pharmaceuticalcomposition which can be employed in one of the uses according to theinvention.

The use of suitable antisense nucleic acids is further described e.g. inZheng and Kemeny (1995) Clin. Exp. Immunol., 100, 380-2; Nellen andLichtenstein (1993) Trends Biochem. Sci., 18, 419-23, Stein (1992)Leukemia, 6, 697-74 or Yacyshyn, B. R. et al. (1998) Gastroenterology,114, 1142).

The production and use of siRNAs as tools for RNA interference in theprocess to down regulate or to switch off gene expression, here PAK geneexpression, is e.g. described in Elbashir, S. M. et al. (2001) GenesDev., 15, 188 or Elbashir, S. M. et al. (2001) Nature, 411, 494.

Ribozymes are also suitable tools to inhibit the translation of nucleicacids, here the RAK gene, because they are able to specifically bind andcut the mRNAs. They are e.g. described in Amarzguioui et al. (1998)Cell. Mol. Life Sci., 54, 1175-202; Vaish et al. (1998) Nucleic AcidsRes., 26, 5237-42; Persidis (1997) Nat. Biotechnol., 15, 921-2 orCouture and Stinchcomb (1996) Trends Genet., 12, 510-5.

Thus, the nucleic acids described can be used to inhibit or reduce theexpression of the PAK genes in the cells both in vivo and in vitro andconsequently act as a PAK inhibitor in the sense of the presentinvention. A single-stranded DNA or RNA is preferred for the use as anantisense oligonucleotide or ribozyme, respectively. For the productionof the medicament the PAK inhibitors of the present invention areusually formulated with one or more pharmaceutically acceptableadditives or auxiliary substances, such as physiological buffersolution, e.g. sodium chloride solution, demineralized water,stabilizers, such as protease or nuclease inhibitors, preferablyaprotinin, ε-aminocaproic acid or pepstatin A or sequestering agentssuch as EDTA, gel formulations, such as white vaseline, low-viscosityparaffin and/or yellow wax, etc. depending on the kind ofadministration.

Suitable further additives are, for example, detergents, such as, forexample, Triton X-100 or sodium deoxycholate, but also polyols, such as,for example, polyethylene glycol or glycerol, sugars, such as, forexample, sucrose or glucose, zwifterionic compounds, such as, forexample, amino acids such as glycine or in particular taurine or betaineand/or a protein, such as, for example, bovine or human serum albumin.Detergents, polyols and/or zwitterionic compounds are preferred.

The physiological buffer solution preferably has a pH of approx.6.0-8.0, expecially a pH of approx. 6.8-7.8, in particular a pH ofapprox. 7.4, and/or an osmolarity of approx. 200-400 milliosmol/liter,preferably of approx. 290-310 milliosmol/liter. The pH of the medicamentis in general adjusted using a suitable organic or inorganic buffer,such as, for example, preferably using a phosphate buffer, tris buffer(tris(hydroxymethyl)aminomethane), HEPES buffer([4-(2-hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer(3-morpholino-1-propanesulphonic acid). The choice of the respectivebuffer in general depends on the desired buffer molarity. Phosphatebuffer is suitable, for example, for injection and infusion solutions.

The medicament can be administered in a conventional manner, e.g. bymeans of oral dosage forms, such as, for example, tablets or capsules,by means of the mucous membranes, for example the nose or the oralcavity, in the form of dispositories implanted under the skin, by meansof injections, infusions or gels which contain the medicaments accordingto the invention. It is further possible to administer the medicamenttopically and locally in order to treat the particular joint disease asdescribed above, if appropriate, in the form of liposome complexes.Furthermore, the treatment can be carried out by means of a transdermaltherapeutic system (TTS), which makes possible a temporally controlledrelease of the medicaments. TTS are known for example, from EP 0 944 398A1, EP 0 916 336 A1, EP 0 889 723 A1 or EP 0 852 493 A1.

Injection solutions are in general used if only relatively small amountsof a solution or suspension, for example about 1 to about 20 ml, are tobe administered to the body. Infusion solutions are in general used if alarger amount of a solution or suspension, for example one or morelitres, are to be administered. Since, in contrast to the infusionsolution, only a few millilitres are administered in the case ofinjection solutions, small differences from the pH and from the osmoticpressure of the blood or the tissue fluid in the injection do not makethemselves noticeable or only make themselves noticeable to aninsignificant extent with respect to pain sensation. Dilution of theformulation according to the invention before use is therefore ingeneral not necessary. In the case of the administration of relativelylarge amounts, however, the formulation according to the inventionshould be diluted briefly before administration to such an extent thatan at least approximately isotonic solution is obtained. An example ofan isotonic solution is a 0.9% strength sodium chloride solution. In thecase of infusion, the dilution can be carried out, for example, usingsterile water while the administration can be carried out, for example,via a so-called bypass.

The above-described nucleic acids can be used in naked form, in the formof gene transfer vectors or complexed with liposomes or gold particles.

Examples of gene transfer vectors are viral vectors, for exampleadenoviral vectors or retroviral vectors (Lindemann et al. (1997), Mol.Med., 3, 466-76; Springer et al. (1988) Mol. Cell., 2, 549-58).Complexes with liposomes usually achieve a very high efficiency oftransfection, in particular of skin cells (Alexander and Akhurst, 1995,Hum. Mol. Genet. 4:2279-85). In lipofection, small, unilamellar vesiclescomposed of cationic lipids are prepared by ultrasonicating the liposomesuspension. The DNA is bound ionically on the surface of the liposomesin a ratio which is such that a positive net charge remains and all theplasmid DNA is complexed by the liposomes. In addition to the DOTMA(1,2-dioleyloxypropyl-3-trimethylammonium bromide) and DOPE(dioleoylphosphatidylethanolamine) lipid mixtures employed by Feigner,P. L. et al. (1987), Proc. Natl. Acad. Sci USA, 84, 7413-7414, a largenumber of lipid formulations have by now been synthesized and tested fortheir efficiency in transfecting a variety of cell lines (Behr et al.(1989) Proc. Natl. Acad. Sci. USA, 86, 6982-6986; Gao and Huang (1991),Biochim. Biophys. Acta, 1189, 195-203; Felgner et al. (1994) J. Biol.Chem., 269, 2550-2561). Examples of the lipid formulations are DOTAPN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate orDOGS (dioctadecylamidoglycylspermine).

Auxiliary substances which increase the transfer of nucleic acids intothe cell can, for example, be proteins or peptides which are bound tothe DNA or synthetic peptide-DNA molecules which enable the nucleic acidto be transported into the nucleus of the cell (Schwartz et al. (1999)Gene Therapy 6:282; Branden et al. (1999) Nature Biotech., 17, 784).Auxiliary substances also include molecules which enable nucleic acidsto be released into the cytoplasm of the cell (Planck et al. (1994) J.Biol. Chem., 269, 12918; Kichler et al. (1997) Bioconj. Chem., 8, 213)or, for example liposomes (Uhlmann and Peyman (1990), supra).

Another, particularly suitable form can be obtained by applying theabove-described nucleic acids to gold particles and firing theseparticles into tissue or cells using what is termed a “gene gun” (Wanget al. (1999) J. Invest. Dermatol. 112:775-81, Tuting et al. (1998) J.Invest. Dermatol. 111:183-8).

Another subject matter of the present invention is the use of PAK or thePAK gene as a target for the discovery of a PAK inhibitor for thetreatment of a joint disease, in particular a degenerative joint diseasesuch as osteoarthritis or an inflammatory joint disease such asrheumatoid arthritis, and/or for the treatment of joint pain, inparticular by reducing the joint pain in degenerative joint diseases.Preferably the PAK inhibitor can be used in form of a medicament asdescribed above.

Accordingly, the present invention refers also to a method of screeninga PAK inhibitor, wherein the method comprises the steps of:

-   (a) providing PAK or the PAK gene,-   (b) providing a test compound, and-   (c) measuring or detecting the influence of the test compound on PAK    or the PAK gene.

In general, PAK or the PAK gene is provided e.g. in an assay system andbrought directly or indirectly into contact with a test compound, inparticular a biochemical or chemical test compound, e.g. in the form ofa chemical compound library. Then, the influence of the test compound onPAK or the PAK gene is measured or detected. Thereafter, suitableinhibitors can be analyzed and/or isolated. For the screening ofchemical compound libraries, the use of high-throughput assays arepreferred which are known to the skilled person or which arecommercially available.

According to the present invention the term “chemical compound library”refers to a plurality of chemical compounds that have been assembledfrom any of multiple sources, including chemically synthesized moleculesand natural products, or that have been generated by combinatorialchemistry techniques.

In general, the influence of the test compound on PAK or the PAK gene ismeasured or detected in a heterogeneous or homogeneous assay. As usedherein, a heterogeneous assay is an assay which includes one or morewashing steps, whereas in a homogeneous assay such washing steps are notnecessary. The reagents and compounds are only mixed and measured.

Suitable functional assays may be based on the gene expression of PAK,the direct activation of PAK by GTPases such as Cdc42, Rac1, Wrch-1 orChp or the complex formation with activated (GTP-bound) p21. In thepresence of a biochemical or chemical compound to be tested as aninhibitor of PAK the gene expression, the direct activation or thecomplex formation with other proteins, e.g. cellular proteins as e.g.specified above, can be measures by means generally known to a skilledperson. In general, commercially available kinase assays systemsquantitatively detect the amount of phosphate incorporated in asubstrate.

For example, the prevention of formation of peripheral actin microspikesand associated loss of stress fibers can be measured as described inZhao, Z. S. et al. (1998), supra.

Heterogeneous assays are, for example, ELISA, DELFIA, SPA and flashplateassays.

ELISA (enzyme linked immuno sorbent assay)-based assays are offered byvarious companies. The assays employ random peptides that can bephosphorylated by a kinase, such as PAK. Kinase-containing samples areusually diluted into a reaction buffer containing e.g. ATP and requisitecations and then added to plate wells. Reactions are stopped by simplyremoving the mixtures. Thereafter, the plates are washed. The reactionis initiated e.g. by the addition of a biotinylated substrate to thekinase. After the reaction, a specific antibody is added. The samplesare usually transferred to pre-blocked protein-G plates and afterwashing e.g streptavidin-HRP is added. Thereafter, unboundstreptavidin-HRP (horseradish peroxidase) is removed, the peroxidasecolour reaction is initiated by addition of the peroxidase substrate andthe optical density is measured in a suitable densitometer.

DELFIA (dissociation enhanced lanthanide fluoro immuno assay)-basedassays are solid phase assay. The antibody is usually labelled withEuropium or another lanthanide and the Europium fluorescence is detectedafter having washed away unbound Europium-labelled antibodies.

SPA (scintillation proximity assay) and the flashplate assay usuallyexploit biotin/avidin interactions for capturing radiolabelledsubstrates. Generally the reaction mixture includes the kinase, abiotinylated peptide substrate and γ-[P³³]ATP. After the reaction, thebiotinylated peptides are captured by streptavidin. In the SPAdetection, streptavidin is bound on scintillant containing beads whereasin the flashplate detection, streptavidin is bound to the interior ofthe well of scintillant containing microplates. Once immobilized, theradiolabelled substrate is close enough to the scintillant to stimulatethe emission of light.

Alternative homogeneous assays are, for example, TR-FRET, FP, ALPHA andgene assays.

TR-FRET (time-resolved fluorescence resonance energy transfer)-basedassays are assays which usually exploit the fluorescence resonanceenergy transfer between Europium and APC, a modified allophycocyanin orother dyes with overlapping spectra such as Cy3/Cy5 or Cy5/Cy7 (Schobel,U. et al. (1999) Bioconjugate Chem. 10, 1107-1114). After excitatione.g. of Europium with light at 337 nm, the molecule fluoresces at 620nm. But if this fluorophore is close enough to APC, the Europium willtransfer its excitation energy to APC, which fluoresces at 665 nm. Thekinase substrate is usually a biotin-labelled substrate. After thekinase reaction, Europium-labelled-(P)-specific antibodies are addedalong with streptavidin-APC. The phosphorylated peptides bring theEuropium-labelled antibody and the streptavidin-APC into close contact.The close proximity of the APC to the Europium fluorophore will cause aquenching of the Europium fluorescence at benefit of the APCfluorescence (FRET).

Fluorescence polarisation (FP)-based assays are assays which usepolarized light to excite fluorescent substrate peptides in solution.These fluorescent peptides are free in solution and tumble, causing theemitted light to become depolarised. When the substrate peptide binds toa larger molecule, however, such as (P)-Tyr, its tumbling rates aregreatly decreased, and the emitted light remains highly polarized. For akinase assay there are generally two options:

(a) A fluorescent phosphopeptide tracer is bound to a (P)-specificantibody. Phosphorylated products will compete the fluorescentphosphopeptide from the antibody resulting in a change of thepolarisation from high to low.

(b) A phosphorylated substrate peptide binds to the phosphospecificantibody resulting in a change of polarisation from low to high.

ALPHA (amplified luminescent proximity homogenous)-based assays, areassays which rely on the transfer of singlet oxygen between donor andacceptor beads brought into proximity by a phosphorylated peptide. Uponexcitation at 680 nm, photosensitisers in donor beads convert ambientoxygen to singlet-state oxygen, which diffuses up to a distance of 200nm. Chemiluminescent groups in the acceptor beads transfer energy tofluorescent acceptors within the bead, which then emits light atapproximately 600 nm.

EFC (enzyme fragment complementation)-based assays or equivalent assayscan be used in particular for high-throughput screening of compounds.The EFC assay is based on an engineered β-galactosidase enzyme thatconsists of two fragments—the enzyme acceptor (EA) and the enzyme donor(ED). When the fragments are separated, there is no β-galactosidaseactivity, but when the fragments are together they associate(complement) to form active enzyme. The EFC assay utilizes an ED-analyteconjugate in which the analyte may be recognized by a specific bindingprotein, such as an antibody or receptor. In the absence of the specificbinding protein, the ED-analyte conjugate is capable of complementing EAto form active β-galactosidase, producing a positive luminescent signal.If the ED-analyte conjugate is bound by a specific binding protein,complementation with EA is prevented, and there is no signal. If freeanalyte is provided (in a sample), it will compete with the ED-analyteconjugate for binding to the specific binding protein. Free analyte willrelease ED-analyte conjugate for complementation with EA, producing asignal dependent upon the amount of free analyte present in the sample.

An example of a gene assay is the two-hybrid system assay (Fields andSternglanz (1994) Trends in Genetics, 10, 286-292; Colas and Brent(1998) TIBTECH, 16, 355-363). In this test, cells are transformed withexpression vectors which are expressing fusion proteins consisting ofthe polypeptide according to the invention and a DNA-binding domain of atranscription factor such as Gal4 or LexA. The transformed cellsadditionally contain a reporter gene whose promoter contains bindingsites for the corresponding DNA-binding domain. By transforming withanother expression vector which is expressing a second fusion proteinconsisting of a known or unknown polypeptide and an activation domain,for example from Gal4 or herpes simplex virus VP16, the expression ofthe reporter gene can be greatly increased if the second fusion proteininteracts with the polypeptide. Consequently this test system can beused for screening for biochemical or chemical compounds which inhibitan interaction between PAK and e.g. GTPases, such as Cdc42, Rac1, Wrch-1or Chp, or activated (GTP-bound) p21 (see e.g. Vidal and Endoh (1999)Trends in Biotechnology, 17, 374-81). In this way, it is possiblerapidly to identify novel active compounds which can be used for thetreatment of joint diseases.

Alternatively, in case the test compound is a protein or peptide,coexpression of this test compound with a GTPase, such as Rac1, Wrch-1,Chp or the constitutively-active Cdc42, as an activator of PAK in thepresence of PAK can be used to measure or detect the influence of thetest compound on PAK as e.g. described in Zhao, Z. S. et al. (1998),supra).

Another example of a gene assay is a functional assay wherein theactivity of the kinase is converted into a functional cellular responsesuch as growth, growth arrest, differentiation or apoptosis. For thistype of screening yeast is a particularly suitable model system. Forexample in a PAK 1-yeast functional assay, when cultured on glucosecontaining medium, the e.g. PAK 1-yeast cells grow like normal yeastcells. When, however, being exposed to galactose, the intracellularexpression of PAK 1 is induced causing the yeast cell to die. Compoundsthat inhibit PAK 1 activity prevent the cell death in this case.

Another assay is based on solid phase-bound polypeptides such as PAK,GTPases, such as Cdc42, Rac1, Wrch-1 or Chp, or activated (GRP-bound)p21 and the interference with the compounds to be tested. Thus, a testcompound, for example, contain a detectable marker, for example, thecompound can be radioactively labelled, fluorescence-labelled orluminescence-labelled as already explained above. Furthermore, compoundscan be coupled to proteins which permit indirect detection, for exampleby means of enzymatic catalysis employing a peroxidase assay which usesa chromogenic substrate or by means of binding a detectable antibody.Another possibility is that of investigating the solid phase-boundprotein complexes by means of mass spectrometry (SELDI). Changes in theconformation of e.g. PAC or the other proteins described above as theresult of interaction with a test substance can be detected, forexample, by the change in the fluorescence of an endogenous tryptophanresidue in the polypeptide.

The solid phase-bound polypeptides can also be part of an array. Methodsfor preparing such arrays using solid phase chemistry and photolabileprotecting groups are disclosed, for example, in U.S. Pat. No.5,744,305. These arrays can also be brought into contact with testcompound or compound libraries and tested for interaction, for examplebinding or changing conformation.

In another embodiment of the present invention, the method is carriedout using whole cells. Usually cells growing at the bottom of multiwellplates are fixed and permeabilized, blocked and incubated with e.g. aprimary (P)-specific antibody against the substrate of interest. Then,e.g. Europium labelled or HRP conjugated secondary antibodies inconjunction with specific chemiluminescent or calorimetric substances,e.g. as described above, are utilized to generate the signal. Incombination with the use of a microscope not only the amount of(P)-specific antibodies can be quantified on the single cell level, butalso phosphorylation-induced translocations of a substrate ormorphological changes of the cells.

Advantageously the method of the present invention is carried out in arobotics system e.g. including robotic plating and a robotic liquidtransfer system, e.g. using microfluidics, i.e. channeled structured.

In another embodiment of the present invention, the method is carriedout in form of a high-through put screening system. In such a systemadvantageously the screening method is automated and miniaturized, inparticular it uses miniaturized wells and microfluidics controlled by aroboter.

In another embodiment the present invention refers also to a method forproducing a medicament for the treatment of a joint disease, inparticular a degenerative joint disease such as osteoarthritis or aninflammatory joint disease such as rheumatoid arthritis, and/or for thetreatment of a joint pain, in particular by reducing the joint pain indegenerative joint diseases, wherein the method comprises the steps of:

-   (a) carrying out the method according to any of the claims 13 to 19,-   (b) isolating a measured or detected test compound suitable for the    treatment of a joint disease and/or a joint pain, and-   (c) formulating the measured or detected test compounds with one or    more pharmaceutically acceptable carriers or auxiliary substances,    e.g. as described above.

The following Figures, Sequences and Examples shall explain the presentinvention without limiting the scope of the invention.

DESCRIPTION OF THE FIGURES

FIGS. 1A-C show the Expression of PAK1 in extracts of HEK293 cells andin human primary chondrocytes.

Extracts of human HEK293 cells as well as human primary chondrocytesoriginating from cartilage of osteoarthritis patients and controlcartilage were separated by 2-dimensional gel electrophoresis. Proteinswere transferred to PVDF membranes by Western blotting and PAK1 wasdetected by a specific antibody.

FIG. 2 shows that the overexpression of the PAK1-ID repressed the IL-1βinduced expression of MMP13 in human SW1353 cells.

The PAK1 inhibitory domain was subcloned into the pCEP4 vector andtransfected into SW1353 cells. Where indicated, IL-1β was used as astimulus at a concentration of 10 ng/ml for 24 h. Supernatants werecollected and the amount of MMP13 protein was determined by ELISA.Values are shown in arbitrary units and represent at least 3 independentresults ±SD. The chequered column represents the experiments with thevector pCEP4 (void vector) and the black column represents theexperiments with the vector pCEP4_PAK1-ID.

FIG. 3 shows that overexpression of the PAK1-ID repressed the IL-1βinduced expression of PGE2 in human SW1353 cells.

The PAK1 inhibitory domain was subcloned into the pCEP4 vector andtransfected into SW1353 cells. Cells were stimulated with a combinationof IL-1β and TNFα both at a concentration of 10 ng/ml for 24 h.Supernatants were collected and the amount of MMP13 protein wasdetermined by ELISA. Values in arbitrary units represent the mean of twoexperiments. The punctured column represents the experiments with novector plus IL1β and TNFα. The chequered column represents theexperiments with the vector pCEP4 (void vector) plus IL1β and TNFα. Theblack column represents the experiments with the vector pCEP4_PAK1-IDplus IL1β and TNFα.

FIG. 4 shows that overexpression of the PAK1-ID represses the IL-1βinduced expression of IL-8 in human HEK293 cells.

The PAK1 inhibitory domain subcloned into the pCDNA3.1 vector wastransiently transfected into HEK293 cells. Cells were stimulated withIL-1β at a concentration of 10 ng/ml for the indicated time periods.Supernatants were collected and the amount of IL-8 protein wasdetermined by ELISA. Values in arbitrary units represent the mean of twoexperiments. The circles represents the experiments with the vectorpCDNA3.1_PAK1-ID plus IL-1β and the squares represent the experimentswith the vector pCDNA3.1 (void vector) plus IL-1β.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the nucleic acid sequence of PAK1.

SEQ ID NO: 2 shows the amino acid sequence of PAK1.

SEQ ID NO: 3 shows the amino acid sequence of PAK2.

SEQ ID NO: 4 shows the amino acid sequence of PAK3.

SEQ ID NO: 5 shows the amino acid sequence of PAK-4.

SEQ ID NO: 6 shows the amino acid sequence of the PAK1 inhibitor domain(PAK1-ID).

SEQ ID NO: 7 shows the amino acid sequence of a proline-rich inhibitorsequence of PAK1 according to Kiosses, W. B. (2002), supra.

SEQ ID NO: 8 shows the amino acid sequence of a synthetic peptidecontaining the PAK1 proline-rich sequence fused to the polybasicsequence from the HIV tat protein according to Kiosses, W. B. (2002),supra.

SEQ ID NO: 9 shows a first PCR primer for the targeting of conservedsequences between human and mouse PAK1 cDNAs.

SEQ ID NO: 10 shows a second PCR primer for the targeting of conservedsequences between human and mouse PAK1 cDNAs.

SEQ ID NO: 11 shows a first PCR primer for the amplification of thePAK1-ID.

SEQ ID NO: 12 shows a second PCR primer for the amplification of thePAK1-ID.

EXAMPLES 1. Methods 1.1 Reverse Transcription-Polymerase Chain Reaction(RT-PCR)

Total RNA (1 μg) was first digested with 1 unit of RNase-free DNase I toavoid contamination by genomic DNA. DNase 1-treated RNA was thenreverse-transcribed to cDNA using Thermoscript reverse transcriptase(Life Technologies, Inc.) primed with oligo(dT)20 according to theprocedure supplied by the manufacturer. PCR was performed using5′-TGGCTGGAGGCTCCTTGACA-3′ (SEQ ID NO: 9) and5′-GAGGGCTTGGCAATCTTCAGGA-3′ (SEQ ID NO: 10) as primers (MWG Biotech AG,Germany) to target conserved sequences between human and mouse PAK1cDNAs. PCR conditions were 95° C./30 s, 60° C./30 s and 72° C./45 s for25 cycles using 2.6 units of Expand High Fidelity PCR DNA polymerase(Roche Diagnostics GmbH, Germany) per 50-μl reaction

1.2 Design of Vector Constructs

The polypeptide encoding PAK1 residues 83 to 149 identified asautoinhibitory domain of PAK1 (Zhao, Z. S. et al. (1998), supra) wasamplified by PCR. The primers used for amplification were5′ATCGCCACCATGTACCCTTATGATGTGCCAGATTATGCCCACACAATTCATGTC GGTTTTG-3′ (SEQID NO: 11) (Kozak sequence is underlined, hemagglutinin (HA) tag is inbold) and 5′-ATCTTATGACTTATCTGTAAAGCTCATG-3′ (SEQ ID NO: 12) (MWG,Biotech AG, Germany). PCR conditions were 95° C./30 s, 60° C./30 s and72° C./30 s for 25 cycles using 2.6 units of Expand High Fidelity PCRDNA polymerase (Roche Diagnostics GmbH, Germany) per 50-μl reaction. ThePCR product was subcloned into the pCR-TOPO2.1 vector (Invitrogen GmbH,Germany). The HA tagged-PAK1 was introduced into the HindIII and XbaIsites of the mammalian pcDNA3.1 (Invitrogen GmbH, Germany). The PAK1-IDwas inserted into the pCEP4 plasmid (Invitrogen GmbH, Germany) via a 5′HindIII and a 3′ NotI restriction site. All plasmids were verified bysequencing.

1.3 Cell Culture

Cultures of human SW1353 chondrosarcoma were grown in Dulbecco'smodified Eagles's medium (DMEM) containing 10% fetal calf serum (FCS)and penicillin/streptomycin (37° C., 5% CO2). For transfections,6×104/well were cultured overnight and at the next day transfected with2 μg DNA and 10 μl GenePORTER™ Transfection Reagent (Gene TherapySystems, Inc., San Diego, Calif., U.S.A.). After 3 h, an equal volume ofmedium containing 20% FCS was added and incubated overnight. In the caseof overexpression with the pCEP4 vector (Invitrogen GmbH, Germany), twodays after transfection the cells were selected with 200 μg/mlhygromycin B (Invitrogen GmbH, Germany). The transfection efficiency wasexamined with a FACScan (Becton Dickinson Immunocytometry Systems, Inc.,Mountain View, Calif., U.S.A.) and on an inverted fluorescencemicroscope. For most experiments, 60000 cells were transferred into eachwell of a 35 mm 6-well plate. Prior to stimulation the cells were washedwith phosphate buffered saline (PBS) and cultivated for 30 min in DMEMwithout FCS. For the experiment, the cells were placed for 24 h in 1 mlserum-free DMEM, with or without 10 ng/ml human IL-1β (Roche DiagnosticsGmbH Germany) and with or without 10 ng/ml TNFα (Roche Diagnostics GmbH,Germany). Human embryonic kidney (HEK) 293 were maintained in Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serum. Fortransfection experiments, cells were seeded at 5×10⁵ cells per well into6-well dishes 24 h before transfection. Cells were incubated for 4 h in1.0 ml of serum-free medium containing 20 μl of lipofectAMINE® (LifeTechnologies, Inc., U.S.A.) and 5.0 μg of total DNA per well(pcDNA3.1-PAK1 (83-149) plasmid or pcDNA.1 empty vector used ascontrol). Cells were either left untreated or stimulated withinterleukin-1β (10 ng/ml; R&D Systems, Inc., Minneapolis, Minn., U.S.A.)following a period of recovery (16 h) in medium containing 10% fetalcalf bovine. Culture supernatant were removed and expression of IL-8(R&D Systems, Inc., Minneapolis, Minn., U.S.A.), PGE2 (SpiBiom Inc.),and MMP-13 (Amersham Pharmacia Biotech, Inc.) was monitored by ELISAaccording to the procedure supplied by the manufacturer.

1.4 Western Blot Analysis and ELISA

To assess the expression of the HA-tagged PAK¹⁸³⁻¹⁴⁹ peptides, cellswere lysed into 100 μl of Lysis Buffer (20 mM MOPS, 2 mM EGTA, 5 mMEDTA, 0.5% Nonidet P-40 supplemented by complete EDTA-free proteaseinhibitor cocktail (Roche Diagnostics GmbH, Germany), and proteinconcentration was determined using BCA Protein assay kit (PierceBiotechnology, Inc. Rockford, Ill., U.S.A.). Proteins were separated by4-12% NuPAGE gel (Invitrogen GmbH, Germany) before transfer to PVDFmembrane (Millipore Corp., U.S.A.).

Protein extracts from human primary chondrocytes fromosteoarthritis-affected patient tissue as well as from normal tissuewere prepared according to standard techniques. Expression of PAK1 wasinvestigated by two-dimensional gel electrophoresis and immunoblottingusing 30 μg of proteins. For 2D-gel electrophoresis, isoelectricfocusing was performed using linear 7-cm immobilized pH 3-10 gradientIPG strips (Bio-Rad Laboratories, Inc., U.S.A.) on a protein IEF Cell(Bio-Rad Laboratories, Inc., U.S.A.). The final focusing step was at4000 V for 8 h. The second dimension was performed on NuPAGE Novex 4-12%ZOOM Gel (Invitrogen GmbH, Germany) and the proteins were transferred onPVDF membrane (Millipore Corp., U.S.A.).

For immunoblotting, HA-tagged PAK¹⁸³⁻¹⁴⁹ and PAK1 proteins were detectedusing HA-tag polyclonal antibody (BD Biosciences Clontech, Palo Alto,Calif., U.S.A.) and αPAK(N-20) polyclonal antibody (Santa Cruz),respectively. Briefly, membranes were blocked with TBS-T (150 mM NaCl,20 mM Tris, pH 7.6, 0.1% Tween 209 containing 5% fat-free dried milk for1 h at room temperature; incubated in TBS-T containing either a 1/500dilution of HA-tag antibody or a 1/50 dilution of αPAK(N-20) antibodiesfor 1 h at RT, and finally incubated in TBS-T containing 1/10000anti-rabbit horseradish peroxydase-conjugated antibody (Pierce) for 1 h.Membranes were processed using ECL according to the manufacturer'sinstructions (Amersham Pharmacia Biotech, Inc.).

For the analysis of MMP13, IL-8 and PGE2 protein levels, supernatants ofcell cultures were determined by ELISA for MMP13 (Amersham PharmaciaBiotech, Inc.), IL-8 (R&D Systems, Inc. Minneapolis, Minn., U.S.A.) andPGE2 (SpiBio) as described by the supplier.

2. Results 2.1 PAK1 Expression in Human Chondrosarcoma Cell Lines and inMouse Primary Chondrocytes

Using RT-PCR, the expression of PAK1 in human SW1353 chondrocyte celllines, as well as in mouse primary cells stimulated or non-stimulatedwith retinoic acid were analyzed.

The results obtained in this experiment clearly demonstrate that PAK1 isexpressed in the human SW1353 chondrosarcoma cell line, as well as inmouse primary chondrocytes. In a further set of experiments RT-PCR wasused to confirm expression of PAK1 in the human CH8 chondrocyte cellline.

The results shown on RNA level have been confirmed also on protein levelby using Western blot analysis with a specific antibody for PAK1 (FIG.1).

Taken together, the results on RNA and protein level confirm that PAK1is expressed in human and mouse chondrocyte cell lines and primarychondrocytes.

2.2 Interference of the Expression of the Inhibitory Domain of PAK1 withIL-1 Signalling:

MMP13 (matrix metalloproteinase 13) represents an important markerprotein for the IL-1 induced expression of proteins that are relevantfor osteoarthritis. The PAK1 inhibitory domain (PAK1-ID) in the humanchondrosarcoma cell line SW1353 was overexpressed in order to study therequirement for PAK1 for transmission of the signal that leads from IL-1to the induction of MMP13 expression (FIG. 2).

In transfected as well as in non-transfected SW1353 cells, exposure toIL-1β led to a large increase in the expression and release of MMP13into the medium. The results of this experiment clearly demonstratedthat overexpression of the PAK1-ID inhibits the IL-1β-induced expressionof MMP13 in the human SW1353 chondrocyte cell line by ˜55%. Furthermore,a basal level of MMP13 secretion into the medium could be observed intransfected as well as in non-transfected SW1353 cells. Expression ofthe PAK1-ID can inhibit the IL-1β-induced expression of MMP13 in thesenon-stimulated cells by >80%. Taken together, the results confirm thatPAK1 is required for the IL-1β induced expression of MMP13.

Another important marker gene is the prostaglandine PGE2. The regulationof PGE2 expression in SW1353 cells was analyzed in the presence andabsence, respectively, of the PAK1-ID (FIG. 3).

Like seen for the inhibition of MMP13 expression, these resultsconfirmed that inhibition of PAK1 through overexpression of the PAK1-IDleads to a significant down-regulation of the IL-1β induced signallingpathways. IL-1β induced expression of PGE2 was inhibited by more than50% in the SW1353 cell line. Furthermore, regulation of PGE2 indicatesan involvement for PAK1 in the processes that are associated with painin osteoarthritis.

In addition to it's effect in SW1353 cells we investigated the effect ofthe PAK1-ID, i.e. the interference with PAK1 activity by it's biologicalinhibitor also in the human embryonic kidney HEK293 cell line (FIG. 4).

The result of this experiment confirmed the findings obtained in thestudies in SW1353 cells. PAK1 plays an essential role in the regulationof the signalling cascade that are induced by IL-1β and lead from theactivation of the IL-1R down to the activation of transcription factorsupregulating the expression of marker genes that are relevant forosteoarthritis and pain such as MMP13, IL-8, and PGE2.

1. A method of screening a PAK inhibitor, wherein the method comprises: (a) providing PAK or the PAK gene, (b) providing a test compound, and (c) measuring or detecting the influence of the test compound on PAK or the PAK gene.
 2. The method according to claim 1, wherein the test compound is provided in the form of a chemical compound library.
 3. The method according to claim 1, wherein the influence of the test compound on PAK or the PAK gene is measured or detected in a heterogeneous or homogeneous assay.
 4. The method according to claim 3, wherein the heterogeneous assay is selected from the group consisting of: an ELISA (enzyme linked immuno sorbent assay), a DELFIA (dissociation enhanced lanthanide fluoro immuno assay), an SPA (scintillation proximity assay) and a flashplate assay.
 5. The method according to claim 3, wherein the homogeneous assay is a TR-FRET (time-resolved fluorescence resonance energy transfer) assay, a FP (fluorescence polarisation) assay, an ALPHA (amplified luminescent proximity homogenous assay), an EFC (enzyme fragment complementation) assay or a gene assay.
 6. The method according to claim 1, wherein the method is carried out on an array.
 7. The method according to claim 1, wherein the method is carried out using whole cells.
 8. The method according to claim 1, wherein the method is carried out in a robotics system.
 9. The method according to claim 1, wherein the method is carried out using microfluidics.
 10. The method according to claim 1, wherein the method is a method of high-through put screening of a PAK inhibitor.
 11. The method of claim 1 wherein said PAK is human PAK1.
 12. The method of claim 11 wherein said PAK is human PAK1 of SEQ ID NO:
 2. 13. A method for producing a medicament for the treatment of a joint disease and/or a joint pain, wherein the method comprises the steps of: (a) carrying out the method according to claim 1, (b) isolating a measured or detected test compound suitable for the treatment of a joint disease and/or a joint pain, and (c) formulating the measured or detected test compounds with one or more pharmaceutically acceptable carriers or auxiliary substances. 